Name: using terminal transferase-mediated dutp nick end-labelling (tunel) and caspase 3/7 assays to measure epidermal cell death in frogs with chytridiomycosis
Uploaded: 2018-05-16T13:00:00
Description: Watch this Scientific Journal Video about Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis at JoV

我们感谢下列协助畜牧和收集数据的人: d. Tegtmeier、c. Fossen、波斯富、m. 麦克威廉姆斯、l. Bertola、斯图尔特、n 哈尼和 t Knavel;和 m. Merces 协助解剖。我们还要感谢 m. 麦克法登、哈洛和塔隆加动物园提高低压阿尔宾娜和 g. Marantelli 以提高 P. corroboree。我们感谢 f. Pasmans, a. 马特尔关于细胞凋亡化验的建议, 康斯坦丁, Kladnik 和韦伯的帮助与隧洞化验, t Emeto 和 W. Weßels 的帮助与协议和试剂盒 caspase 3/7 化验。此手稿和协议从 Brannelly et 2017对等 J22中改编而成.

詹姆斯库克大学批准了应用程序中的动物伦理学 A1875 corroboree以及低压阿尔宾娜的 A1897 和 A2171。
1. 畜牧业和监测
<ol><li>在适宜于该物种的环境中单独饲养动物, 并提供适当的水、喂养和清洁计划。每天检查动物。<ol>
<li>使用严重濒危的Pseudophryne corroboree (用于隧洞化验、4节) 和受威胁的Litoria verreauxii 阿尔宾娜(Caspase 3/7 化验, 5 节), 捐赠自圈养育种设...

<ol>
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我们探讨了表皮细胞凋亡和细胞死亡作为一种潜在的病理机制的致命疾病壶菌病或机制的抗病能力的Bd 敏感物种。我们使用两种方法评估表皮细胞死亡, 对原位表皮细胞死亡分析的 caspase 法, 以及在整个感染过程中监测表皮细胞死亡的3/7 项试验。我们发现, 细胞死亡和凋亡与感染负荷相关, 细胞死亡在感染部位明显增高。早期感染时, 表皮细胞凋亡减少, 但随着感染组织内病原菌负担的...

两栖动物目前正在经历一个最大的损失全球生物多样性的任何脊椎动物分类1。这些下降的主要原因是致命的皮肤病壶菌病, 由真菌病原体 Batrachochytrium dendrobatidis, Bd 2 引起.病原体表面上感染表皮, 这可能导致皮肤功能的破坏, 导致严重的电解质丧失, 心跳骤停, 死亡3。目前正在研究针对Bd的各种潜在宿主免疫机制, 例如抗菌肽4、5、皮肤细菌菌群6、免疫细胞受体7、8、和淋巴细胞活动9,10。然而, 很少有研究探讨表皮细胞凋亡和细胞死亡是否是对抗这种致命病原体的免疫机制。
细胞死亡, 无论是通过凋亡 (程序性细胞死亡) 或坏死 (unprogrammed 死亡), 在表皮可能是病理学的Bd感染。先前的研究表明, Bd感染可能诱导细胞凋亡, 因为当皮肤外植体暴露在游动孢子上清液体内 11 中时, 细胞内结的破坏就会被观察到。此外, 在Bd感染的青蛙的变性表皮变化观察使用电子显微镜12,13。Transcriptomic 分析表明, 凋亡通路是上调在感染皮肤14, 两栖脾发生凋亡时, 他们暴露在Bd上清液在体外15。尽管越来越多的证据表明, Bd可以诱导细胞凋亡和宿主细胞死亡体外,在体内研究, 探索或量化凋亡机制, 通过感染的进展缺乏。此外, 不知道宿主是否使用凋亡作为防御性免疫策略来对抗Bd感染, 或者如果凋亡是疾病的病理学。
在本研究中, 我们的目的是通过两种方法来检测感染动物的表皮细胞死亡和细胞凋亡: caspase 3/7 蛋白测定, 以及末端转移酶介导的 dUTP 尼克末端标签 (隧洞) 原位测定。由于每种检测都检测到细胞死亡的不同方面16, 这些方法一起提供了对细胞死亡所涉及的机制的充分理解, 并确保准确测量效果。caspase 3/7 的分析量化了效应半胱氨酸蛋白酶3和7的活动, 这使得定量的内在和外在的凋亡途径。与此相反, 该方法检测 DNA 碎片, 这是由细胞死亡机制, 包括凋亡, 坏死和 pyroptosis17造成的。我们使用三种不同的皮肤部位: 背部、温特和Pseudophryne corroboree的大腿, 利用该方法对临床感染和未感染动物表皮内的细胞死亡位置进行调查。该方法确定细胞死亡的解剖部位, 并鉴别其在特定表皮层内的位置。然后, 我们使用 caspase 3/7 检测在Litoria verreauxii 阿尔宾娜中对整个8周的感染进行时间序列的细胞凋亡量化。我们从相同的动物两周的脚趾提示样本, 并能够关联病原体感染负荷与 caspase 3/7 活动。

<table>
	<tbody>
		<tr>
			<td>POLARstar Omega</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>Luminescent plate reader</td>
		</tr>
		<tr>
			<td>384 well flat clear bottom plate</td>
			<td>Corning</td>
			<td>3707</td>
			<td></td>
		</tr>
		<tr>
			<td>384 well low flange white flat bottom plate</td>
			<td>Corning</td>
			<td>3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar Bacteriological (Oxoid)</td>
			<td>Fisher</td>
			<td>OXLP0011B</td>
			<td></td>
		</tr>
		<tr>
			<td>Formal-Fixx 10% Neutral Buffered Formalin</td>
			<td>Fisher</td>
			<td>6764254</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactose Broth (Oxoid)</td>
			<td>Fisher</td>
			<td>OXCM0137B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher</td>
			<td>BP328-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricane-S (MS-222)</td>
			<td>Fisher</td>
			<td>NC0872873</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>BP1421-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Invitrogen</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile rayon swab</td>
			<td>Medical Wire &#38; Equipment</td>
			<td>MW-113</td>
			<td></td>
		</tr>
		<tr>
			<td>ApopTag Red In Situ Apoptosis Detection Kit</td>
			<td>Merck Millipore</td>
			<td>S7165</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Bradford reagent</td>
			<td>Pierce</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase Glo&#160; 3/7</td>
			<td>Promega</td>
			<td>G8090</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma Aldrich</td>
			<td>H0887-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>1374248-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin hydrolysate Enzymatic</td>
			<td>Sigma-Aldrich</td>
			<td>G0262</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Saline), pH 7.2 (1X)</td>
			<td>Thermo/Life</td>
			<td>20-012-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepman</td>
			<td>Thermo/Life</td>
			<td>4318930</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Advanced Master Mix</td>
			<td>ThermoFisher</td>
			<td>4444556</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorox bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 Proof, Molecular Grade</td>
			<td>Fisher</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS Axio Scan florescent miscroscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Florescent microscope</td>
		</tr>
		<tr>
			<td>3.2mm stainless steel beads</td>
			<td>BioSpec</td>
			<td>11079132SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer ITSI-3 Chytr (5&#8242;-CCTTGATATAATACAGTGTGCCATATGTC-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Primer 5.8S Chytr (5&#8242;-TCGGTTCTCTAGGCAACAGTTT-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Minor groove binder probe Chytr MGB2(5&#8242;-CGAGTCGAAC-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Rotor-Gene qPCR Instruments</td>
			<td>Qiagen</td>
			<td></td>
			<td>qPCR machine</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5ml</td>
			<td>Fisher</td>
			<td>02-681-372</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture petri plates</td>
			<td>Nunc</td>
			<td>263991</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-beadBeater Zircornia-Silicate Beads, 0.5mm</td>
			<td>BioSpec</td>
			<td>11079105Z</td>
			<td></td>
		</tr>
	</tbody>
</table>

using terminal transferase-mediated dutp nick end-labelling (tunel) and caspase 3/7 assays to measure epidermal cell death in frogs with chytridiomycosis

两栖动物在全球范围内的生物多样性遭受巨大损失, 其中一个主要原因是传染病壶菌病。这种疾病是由真菌病原体Batrachochytrium dendrobatidis (Bd) 引起的, 它感染和扰乱了青蛙表皮;然而, 病理变化没有明确的特征。细胞凋亡 (程序性细胞死亡) 可以被病原体用来损伤宿主组织, 但也可以成为病原体去除抗病的宿主机制。在这项研究中, 我们用两种不同的化验方法来量化感染和未感染动物的表皮细胞死亡: 末端转移酶介导的 dUTP 尼克末端标签 (caspase 3/7)。采用腹壁、背侧和大腿皮肤组织, 观察临床感染动物的表皮细胞中的细胞死亡, 并用荧光显微术比较未感染动物的细胞死亡. 为了确定表皮细胞凋亡水平在感染过程中的变化情况, 我们在8周的时间里去掉脚趾头的样本, 并使用提取的蛋白质 caspase 3/7 化验来量化样品中的活动。然后, 我们将 caspase 3/7 活动与感染负荷相关联。该方法对细胞死亡的局部化有很大的作用就地, 但成本高昂, 且每次取样时间密集。caspase 3/7 法对大样本量和时间过程实验有很高的效率。然而, 由于青蛙脚趾尖端活检是小的, 有有限的提取物可用于样本标准化通过蛋白质量化方法, 如布拉德福德化验。因此, 我们建议通过对脚趾活检的摄影分析来估计皮肤表面积, 以避免在样品标准化过程中消耗提取物。

隧洞试验
感染动物中的细胞比未感染的对照动物多。在感染和控制的动物中, 隧洞阳性细胞的原位位置不同。在控制动物中, 在皮肤和表皮层的低水平上, 有一个均匀分布的隧洞阳性细胞 (参见图 1A), 但在受感染的动物中, 在表皮中的隧洞阳性细胞更频繁 (图1B). 感染部位...

Watch this Scientific Journal Video about Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis at JoV

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

我们用两种方法定量测定青蛙壶菌病的表皮细胞死亡。首先, 我们使用终端转移酶介导的 dUTP 尼克末端标签 (隧洞)的原位组织学, 以确定临床感染和未感染动物之间的差异。其次, 用 caspase 3/7 蛋白分析方法对感染细胞凋亡进行时间序列分析。

用末端转移酶介导的 dUTP 尼克末端标记 (Caspase 3/7) 测定青蛙壶菌病的表皮细胞死亡

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...

The overall goal of this experiment is to observe the affect of amphibian fungal pathogen batrachochytrium dendrobatidis on apoptosis and cell death in the epidermal tissue of frogs. This method can help us understand wildlife disease such as if infection causes cell death or, conversely, if there are immune mechanisms against infection. The main advantage of this technique is that it can be performed on small tissue biopsy samples so that small individuals can be repeatedly sampled throughout the course of infection.To begin this procedure, prepare hydrophilic glass slides with sample tissue as outlined in the text protocol. To deparaffinize the tissue sections, wash the slides three times with xylene for five minutes each in a coplin jar. Next, wash the slides with 100%ethanol two times for five minutes each.After this, wash the slides with 95%ethanol and 70%ethanol for three minutes each. Then, wash the slides once with PBS for five minutes. To prepare experimental and negative control slides, pretreat the tissue by adding freshly diluted proteinase K directly to the slides.Incubate the slides for 10 minutes at room temperature. Then, wash the slides in PBS two times for two minutes each. To prepare positive control slides, pretreat the tissue with DN buffer then incubate the slides at room temperature for five minutes.After this, dissolve DNase one in DN buffer to a final concentration of 0.1 micrograms per milliliter. Add the prepared DNase solution to the slides. Incubate the slides for 15 minutes at room temperature.Then, wash the slides with distilled water in a coplin jar five times for three minutes each. Quench the reaction in 3%hydrogen peroxide in PBS for five minutes at room temperature. Next, wash the slides in PBS two times for two minutes each.Then, add equilibrium buffer to the tissue on the slides. Incubate for 10 seconds at room temperature. Then, tap off the excess liquid around the tissue section.Dilute terminal deoxynucleotidyl transferase enzyme in reaction buffer. Then, add it to the tissue on the experimental and positive control slides. Incubate in a humidified chamber for one hour at 37 degrees Celsius.After this, place the slides in a coplin jar containing the working concentration of stop buffer. Agitate for 15 seconds and then incubate the slides at room temperature for 10 minutes. Then, wash the slides with PBS three times for one minute each and tap off the excess liquid.Next, add the anti-digoxigenin conjugate, prewarmed to room temperature, directly onto the tissue. Incubate the slides for 30 minutes at room temperature in a humidified dark chamber. After the incubation is complete, wash the slides with PBS four times for two minutes each.Tap off any excess liquid. Then, add mounting medium to the slides. Add 15 microliters of DAPI solution to the mounting medium on the slides.Cover each slide with a cover slip and seal with nail polish. Allow the slides to dry in the dark. Using a fluorescent microscope, observe the slides immediately upon completion of the standing process.Take photos at 400 times at random intervals along each skin section. Count DAPI-stained blue cells to ensure at least 100 cells per skin section per animal are visible. Next, count the number of TUNEL-positive cells which appear as red single cells with clear cell edges.To begin, use an inverted light microscope at 40 times to take a photo of each of the frog's toes. Draw lines around the edge of the toe clip and use imaging software to estimate the area within the shape. To evaluate the surface area of the three-dimensional skin sample, multiply the estimated area by pi.To extract proteins from a frozen frog toe sample, place the sample in a 1.5 milliliter screw cap microcentrifuge tube containing 100 microliters of sample buffer and two stainless steel beads. Use a bead beater at maximum speed to lyse the cells. After this, place the tube on ice for three minutes.Then, centrifuge the tube to collect the supernatant. To perform the caspase 3/7 assay in a 384-well plate, add 10 microliters of sample buffer as blank and 10 microliters of protein extract in triplicate. Then, pipette 10 microliters of commercially available caspase 3/7 reagent to the protein extract and blank wells.Then, shake the plate gently for 15 seconds to mix the two reagents. Incubate at room temperature in dark for 30 minutes. Finally, use a luminescent plate reader to measure the luminescence of the sample.In this study, the infected animals depict significantly more TUNEL-positive cells in comparison to control animals as illustrated by red or pink-stained cells. In control animals, there are few TUNEL-positive cells, and they are not concentrated in the epidermis. In infected animals, the TUNEL-positive cells are more frequent in the epidermis.TUNEL-positive cells are more concentrated at and directly adjacent to the sights of infected cells. Also, there are more widespread TUNEL-positive cells over the epidermis and in epidermal layers that are deeper than where the fungal pathogen resided. In infected animals, there are more TUNEL-positive cells in all three skin types with a particularly high level in the thigh and venter skin.In the caspase assay, the infection intensity is positively correlated with caspase 3/7 activity. Additionally, caspase 3/7 activity in infected and uninfected animals differed over time. Caspase 3/7 activity decreased within the first few weeks after inoculation and then increased toward the end of week seven in infected animals.Caspase 3/7 activity remained constant in uninfected individuals. While attempting the procedure for the TUNEL assay, it's important to read the slides on the same day as the assay to avoid loss of sensitivity and to keep the slides in a cool dark place to protect the dyes. After watching this video, you should have a good understanding of how to detect apoptosis and cell death using two different detection methods in small tissue samples like amphibian skin.

Research

JoVE Journal

Immunology and Infection

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Competitive Homing Assays to Study Gut-tropic T Cell Migration

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

科研

免疫与感染

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

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insulin ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

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量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

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肠道细菌侵入肠上皮细胞<em&gt;体外</em&gt;使用垂直扩散室模型，显着增强

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

治疗核黄素血小板制品和紫外线光：对有效性高滴度细菌污染

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

接种<em&gt;冈比亚按蚊</em&gt;珠蚊子诱发和测量黑化免疫反应

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...